Novel Regulation of Neuronal Genes Implicated in Alzheimer
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NOVEL REGULATION OF NEURONAL GENES IMPLICATED IN ALZHEIMER DISEASE BY MICRORNA Justin M. Long Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Program in Medical Neuroscience, Indiana University March 2013 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________ Feng C. Zhou, PhD, Chair __________________ Debomoy K. Lahiri, PhD Doctoral Committee ___________________ Richard Nass, PhD June 19, 2012 ___________________________________ Yansheng Du, PhD ___________________________________ Martin R. Farlow, MD ii ACKNOWLEDGEMENTS I must first thank my research advisor, Dr. Debomoy Lahiri, who has provided invaluable advice, encouragement and mentorship during my tenure in his laboratory. I have learned much from him about the process of conducting vigorous science and the importance of communicating that science to the world. I appreciate the time and resources he has provided me during my development as a budding scientist. Without his professional support, my research training and the work described in this dissertation would not have been possible. I must also thank the other members of my research committee: committee chair Dr. Feng Zhou, Dr. Richard Nass, Dr. Yansheng Du and Dr. Martin Farlow. They have been generous in providing their time and expertise throughout my training. Their mark is clearly evident in this dissertation, as their guidance has continually had a positive impact on the direction of my research. I truly appreciate their input. Several collaborators provided key materials that were critical to the success of my research. I thank Dr. P Hemanchandra Reddy (Oregon Health and Science University, Beaverton, OR) and Dr. Peter Nelson (University of Kentucky, Lexington, KY) for providing brain specimens from their collections, Dr. Jack Rogers (Massachusetts General Hospital, Charlestown, MA) for providing plasmids, and Dr. Robert Vassar (Northwestern University, Chicago, IL) for providing anti-BACE1 antibody 3D5. I also thank Dr. Bob Niculescu for valuable feedback on my research and for providing open access to his real-time PCR instrument. Finally, I also wish to express my gratitude to the various funding sources that supported this work, including National Institutes of Aging and Alzheimer’s Association grants to Dr. Lahiri and travel grants from the Alzheimer’s Association and Dr. Joseph Hingtgen. I would also like to thank all members of Dr. Lahiri’s laboratory who have been instrumental in helping me survive the vagaries of biomedical research. I would like to iii thank Dr. Jason Bailey who showed me the ropes as an early graduate student and taught me many valuable laboratory techniques. I also thank Dr. Balmiki Ray for establishing many research models and assays in the laboratory that contributed significantly to this work and for being willing to help with experiments whenever I was in a pinch. I thank Bryan Maloney for his valuable insight on molecular biology and statistical analysis. I also thank Nipun Chopra and Cindy Morgan for tolerating me as bench mates, helping with experiments when needed and general comedic relief. I finally thank all of these fellow lab members for their continued friendship and collegiality that has made time spent in the lab a pleasure. I thank my family and friends for their ever present interest in my work and continual moral support. I thank my two young kids for never failing to bring a smile to my face and reminding me that life does not revolve around the laboratory. Most importantly, I thank my wife Melissa for her enduring patience during this long journey of a career path that I have chosen. Her unfailing love and support throughout this process has been the crutch on which I continue to lean. iv ABSTRACT Justin M. Long NOVEL REGULATION OF NEURONAL GENES IMPLICATED IN ALZHEIMER DISEASE BY MICRORNA Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ- generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post- transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. v Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD. Feng C. Zhou, PhD, Chair vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... xi LIST OF FIGURES ........................................................................................................ xii LIST OF ABBREVIATIONS ........................................................................................... xv CHAPTER 1: INTRODUCTION ...................................................................................... 1 I. Alzheimer Disease (AD) Background ................................................................... 1 A. Clinical and neuropathological overview ......................................................... 1 B. Molecular mechanisms and genetics .............................................................. 4 C. Support for centrality of amyloid-β to AD etiology ........................................... 6 D. Current therapeutic strategies and limitations ................................................. 7 II. Biology of amyloid-β precursor protein (APP) .................................................... 10 A. Biochemical processing of APP .....................................................................10 B. Physiological functions of APP ......................................................................13 C. Transcriptional and post-transcriptional control of APP expression ...............14 III. Biology of BACE1 .............................................................................................. 16 A. Biochemical and physiological function of BACE1 .........................................16 B. Transcriptional and post-transcriptional control of BACE1 expression ...........17 IV. Targeting APP and BACE1 as a therapeutic strategy ........................................ 19 V. Biology of miRNA ............................................................................................... 21 A. Discovery, historical perspective and nomenclature ......................................21 B. Biogenesis.....................................................................................................23 C. Assembly of microribonucleoprotein complex ................................................24 D. Molecular mechanisms of gene regulation ....................................................28 E. miRNA stability and turnover .........................................................................30 F. miRNA dysregulation in AD and contribution to pathophysiology ...................32 G. Role of miRNA in normal and pathological aging ...........................................33 vii VI. Hypothesis and Aims ......................................................................................... 35 CHAPTER 2: MATERIALS AND METHODS ................................................................ 36 I. Tissue culture techniques .................................................................................. 36 A. Culture and maintenance of continuous human cell lines ..............................36 B. Culture and maintenance of primary human fetal brain cells .........................38 C. Transfection of DNA vectors or RNA oligonucleotides into cell lines and primary cultures ............................................................................................39 D. Harvest and preparation of cell culture lysate for RNA and protein analyses ........................................................................................................44